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Xenopus axis formation: induction of goosecoid by injected Xwnt-8 and activin mRNAs.
Steinbeisser H
,
De Robertis EM
,
Ku M
,
Kessler DS
,
Melton DA
.
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In this study, we compare the effects of three mRNAs-goosecoid, activin and Xwnt-8- that are able to induce partial or complete secondary axes when injected into Xenopus embryos. Xwnt-8 injection produces complete secondary axes including head structures whereas activin and goosecoid injection produce partial secondary axes at high frequency that lack head structures anterior to the auditory vesicle and often lack notochord. Xwnt-8 can activate goosecoid only in the deep marginal zone, i.e., in the region in which this organizer-specific homeobox gene is normally expressed on the dorsal side. Activin B mRNA, however, can turn on goosecoid in all regions of the embryo. We also tested the capacity of these gene products to restore axis formation in embryos in which the cortical rotation was blocked by UV irradiation. Whereas Xwnt-8 gives complete rescue of anterior structures, both goosecoid and activin give partial rescue. Rescued axes including hindbrain structures up to level of the auditory vesicle can be obtained at high frequency even in the absence of notochord structures. The possible functions of Wnt-like and activin-like signals and of the goosecoid homeobox gene, and their order of action in the formation of Spemann's organizer are discussed.
Fig. 1. Goosecoid RNA is induced after injection of Xwnt-8 or
activin B mRNA in UV embryos. 4-cell embryos treated with UV
(lane 2) were injected with Xwnt-8 (lane 3) or activin B mRNA
(lane 4). Lane 1; uninjected embryos. RNA was extracted at stage
10.5 and the equivalents of 2.5 embryos were analyzed by
northern blot hybridization using probes for gsc and fibronectin
(fn). The UV treatment resulted in ventralized embryos with a
dorsoanterior index, DAI, of less than 0.5 as defined by the
method of Kao and Elinson (1988).
Fig. 2. Injected Xwnt-8 mRNA induces
goosecoid RNA exclusively in the
marginal zone. Diagram of the injection
experiment where both blastomeres of
2-cell embryos were injected with
Xwnt-8 mRNA (A). At stage 10.5 an in
situ hybridization for gsc was
performed using uninjected embryos
(B,E) and embryos injected with Xwnt-8
RNA (C,F). Embryos were cut through
the dorsal lip as indicated in (D) and
photographed to show a lateral view
with the collapsed blastocoel at the top
(E,F). Arrowhead indicates the dorsal
lip.
Fig. 3. Different spatial activation of goosecoid after injection of Xwnt-8 and activin B mRNA. 8-cell embryos were injected into animal
(an.) or vegetal blastomeres (veg.) as diagramed in (A) and (E) with activin (C,G) or Xwnt-8 RNA (D,H). Embryos injected at opposite
sides of the marginal zone display two regions of gsc expression (I). UV-treated embryos (J) lack gsc expression and dorsal lips, which
can be restored by an injection of activin mRNA into the marginal zone (K). Note that in K activin mRNA induces gsc both above and
below the blastopore lip, while in normal embryos (B) gsc is expressed exclusively above the dorsal lip. In situ hybridization for gsc was
done using uninjected stage 10.5 embryos (B,F) as controls. B,C,D,I,J,K, vegetal pole view; F,G,H, animal pole view. The position of the
dorsal lip is indicated by arrowheads. The embryo in I has been rendered transparent with a clearing agent.
Fig. 4. Induction of axial structures by
injection of gsc mRNA. (A-F) Rescue of
UV-treated embryos; (G,H) ventral
injections into untreated embryos.
(A) Embryo ventralized by UV (DAI=0);
(B) embryo that was microinjected into two
diagonally opposed blastomeres at the 4-cell
stage with gsc mRNA (0.5 ng per
blastomere) showing the formation of
twinned body axes. (C-E) Twinned embryos
injected as in B, which have been stained for
notochord with MZ-15 antibody showing no
notochords, one notochord and two
notochords, respectively. In this experiment
(n=16), 9 embryos showed two rescued axes,
and of these 6 had two notochords, 2 had
one, and 1 (C) had none. The arrowheads
indicate the anterior end of the embryo (B)
and auditory vesicles (C,D,E,G,H).
(F) Transverse section through a notochordless
rescued axis that was whole-mount
stained with the muscle-specific antibody
12/101; note that somites are fused across
the midline. (G,H) Twinned axes induced by
microinjection of gsc mRNA (0.6 ng/cell)
into two ventral blastomeres at the 4-cell
stage; note the absence of notochord in the
secondary axis in G, despite the presence of
auditory vesicles. nt, neural tube; som,
somites; nc, notochord.
Fig. 5. Rescue of axial structures in UV embryos by activin and gsc mRNA. (B-E) The results of an experiment in which either activin B
(100 pg/cell) or gsc mRNA (150 pg/cell) was injected into three adjacent blastomeres of the ‘C’ tier of UV-ventralized 32-cell embryos.
(A) Experimental design; (B) axis rescued by activin mRNA (DAI=0.4 for uninjected UV embryos, n=78, 24 of the injected embryos
showed axial structures, n=28, average DAI=1.5). (C) Axis rescued by gsc RNA (DAI=0.24 for uninjected UV-treated controls, n=104,
12 of the injected embryos showed axial structures, n=22, average DAI=1.1). (D,E) Histological sections of embryos rescued with activin
(D) or gsc (E) mRNA, note that in both cases the axis extends up to the level of the auditory vesicle (av).
